Sickle cell disease (SCD) is the most common life-threatening monogenic disorder in the world with statistics indicating that approximately 80% (230,000) of children affected globally are born in sub-Saharan Africa (Modell and Darlison 2008). SCD is a severe hemoglobinopathy that produces multisystem complications due to the expression of abnormal sickle hemoglobin (HbS). The most common type of SCD is sickle cell anemia (SCA) (also referred to as HbSS or SS disease or hemoglobin S) in which there is homozygosity for the mutation that causes HbS. The more rare types of SCD in which there is heterozygosity (one copy of the mutation that causes HbS and one copy for another abnormal hemoglobin allele) for the mutation include sickle-hemoglobin C (HbSC), sickle β+ thalassemia (HbS/β+) and sickle β0 thalassemia)(HbS/β0).
Sickle cell disease (SCD) can arise from a single point mutation that causes erythrocyte deformation or sickle-shaped erythrocytes (Ingram 1957). Sickled-shaped erythrocytes are associated with clinical manifestations of SCD, such as anemia, recurrent painful vaso-occlusive episodes, infections, acute chest syndrome, pulmonary hypertension, stroke, priapism, osteonecrosis, renal insufficiency, leg ulcers, retinopathies, and cardiac disease (Frenette and Atweh 2007, Steinberg 2008).
Pathophysiology
Sickle-Shaped Erythrocytes
SCD arises from a single point mutation (GAG>GTG) in codon 6 of the HBB globin gene. This point mutation (GAG>GTG) results in the polar hydrophilic molecule, glutamic acid being substituted with the non-polar hydrophobic molecule, valine (β6 Glu→Val) (Ingram 1957). The deoxygenated venous circulation causes the hydrophobic valine residue to associate with hydrophobic regions of adjacent molecules (Mozzarelli, et al 1987). This process of self-assembly (polymerization) generates the sickled hemoglobin molecule (HbS) that damages the membrane and cytoskeleton of the erythrocyte. The HbS repetitively enter into sickling and unsickling cycles incrementally increasing the damage to the erythrocyte membrane (Ischemia-reperfusion (IR) injury) resulting in irreversibly sickle-shaped erythrocytes (Barabino, et al 2010).
C-reactive protein (CRP) (Nath, et al 2005) and the markers of oxidative stress, such as xanthine oxidase (XO), salicylate hydroxylation and expired ethane, are significantly increased following IR injury (Huang, et al 2007, Osarogiagbon, et al 2000). The activation of XO and reduced Tetrahydrobiopterin (BH4) levels in endothelial cells leads to increased reactive oxygen species (ROS) formation, which in turn leads to endothelial nitric oxide synthase (eNOS) uncoupling and further production of superoxide (Thomas, et al 2010). ROS causes endothelial cell membrane lipid peroxidation, inactivation of nitric oxide (NO), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB) and Src/MAP kinase signaling, and induces production of tissue factor (TF), interleukin 8 (IL-8), and surface adhesion molecule expression (Belcher, et al 2006, Radi, et al 1991, Thomas, et al 2010). Thus, the ensuing oxidative stress contributes to hemolysis, inactivation of NO, and erythrocyte, leukocyte and platelet adhesive properties (Kaul, et al 2004, Sultana, et al 1998, Vilas-Boas, et al 2010).
The Sickled-shaped erythrocytes together with endothelial cells, activated leukocytes, platelets and plasma proteins participate in the multistep vaso-occlusion process (Frenette 2002).
TABLE 1Ischemia-reperfusion (IR) injury-relatedmolecules in individuals with SCDExpression statusIR injury-related moleculesin SCDReferencesXanthine oxidase (XO)Up(Osarogiagbon, et al2000)Tetrahydrobiopterin (BH4)Down(Thomas, et al 2010)C-reactive protein (CRP)Up(Nath, et al 2005)Reactive Oxygen SpeciesUp(Huang, et al 2007)(ROS)Endothelial Cells
Vascular homeostasis is harmonized by the endothelial cell vasoregulators regulating blood flow, growth of vascular smooth muscle cells and local inflammation (Huang, et al 2006). Endothelial cells are the primary producers of the major vasodilator, nitric oxide (NO) and prostacyclin, as well as vasoconstrictors such as endothelin, angiotensin II and prostaglandins (Galley and Webster 2004). These endothelial cell vasoregulators are characteristically imbalanced in individuals with SCD, resulting in endothelial dysfunction that contributes to vaso-occlusion process.
SCD is characterized by a reduced bioavailability of NO, due to 1/cell-free plasma hemoglobin and increased arginase activity encouraging hemolysis-related scavenging of NO (Reiter and Gladwin 2003), and 2/eNOS function being diminished due to a decrease in the nitric oxide synthase (NOS) substrate, arginine's ability to dimerize thereby contributing to reduced NO synthesis (Lin, et al 2011). The reduced NO decreases the NO-dependent vaso-dilation, contributing to an increase in vasoconstrictors such as endothelin-1 (ET-1) (Ergul, et al 2004, Werdehoff, et al 1998) and endothelin-3 (ET-3) (Makis, et al 2004). It has been demonstrated that Endothelin-3 induces endothelial cell interleukin 6 (IL-6) expression thereby mediating inflammation. Thus, IL-6 is also characteristically found to be increased in SCD patients (Makis, et al 2004).
TABLE 2Endothelial cell vasoregulators thatis imbalanced in individuals with SCDExpressionEndothelial CellType ofstatus inVasoregulatorsVasoregulatorSCDReferencesNitric Oxidevasodilatordown(Eberhardt,et al 2003)Endothelin-1vasoconstrictorup(Werdehoff,et al 1998)Endothelin-3vasoconstrictorup(Makis,et al 2004)
Activators of endothelial cells include NFκB (Belcher, et al 2005), hypoxia-inducible factor-1 (HIF-1)(Kim, et al 2006), ET-1 (Phelan, et al 1995) and TF (Solovey, et al 1998). Interestingly, it has been demonstrated that eNOS modulates the expression of TF by down-regulates it, however, eNOS is characteristically down-regulated in SCD patients (Solovey, et al 2010). Once activated, the endothelium produces cytokines such as tumour necrosis factor alpha (TNFα) (Lanaro, et al 2009) and interleukin 1 beta (IL-1β) (Croizat 1994, Wanderer 2009), chemokines and/or inflammatory molecules such as granulocyte macrophage-colony stimulating factor (GM-CSF) (Conran, et al 2007), IL-8 (Lanaro, et al 2009), IL-6(Croizat 1994), interleukin 3 (IL-3) (Croizat 1994), interleukin 4 (IL-4) (Musa, et al 2010), and platelet activating factor (PAF) (Oh, et al 1997), and adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule 1 (ICAM-1), selectin E (SELE) and selectin P (SELP) (Chiu, et al 2004, Solovey, et al 1997). Rajan et al. demonstrated that NFκB is required for TNFα-induced expression of VCAM-1, ICAM-1 and SELE in endothelial cells (Rajan, et al 2008). The production of these inflammatory mediators and cell adhesion molecules is perpetuated by TNFα and IL-1β being potent activators of the endothelium (Segers, et al 2006). Heme oxygenase-1 (HO-1) (Belcher, et al 2006) and interleukin 10 (IL-10) (Musa, et al 2010) are characteristically found to be increased in SCD patients in an attempt to counteract the induced inflammation. HO-1 breaks down heme released during hemolysis thereby limiting oxidative stress and inflammation (Otterbein, et al 2003), whilst IL-10 limits the production of the pro-inflammatory cytokines (Lanaro, et al 2009, Taylor, et al 2001).
TABLE 3Endothelial cell activation-relatedmolecules in individuals with SCDExpressionstatus inMoleculesTypes of MoleculesSCDReferencesNFκBActivator ofup(Belcher, et al 2005)endothelial cellsET-1Activator ofup(Phelan, et al 1995)endothelial cellsHIF-1Activator ofup(Kim, et al 2006)endothelial cellsTFActivator ofup(Solovey, et al 1998)endothelial cellseNOSInhibitor ofdown(Solovey, et al 2010)endothelial cellactivationTNFαcytokine/up(Lanaro, et al 2009)inflammatoryIL-1βcytokine/up(Wanderer 2009)inflammatoryIL-8chemokine/up(Lanaro, et al 2009)inflammatoryIL-4chemokineup(Musa, et al 2010)PAFchemokineup(Oh, et al 1997)IL-6chemokine/up(Croizat 1994)inflammatoryGM-CSFinflammatoryup(Conran, et al 2007)IL-3inflammatoryup(Croizat 1994)VCAM-1adhesionup(Solovey, et al 1997)ICAM-1adhesionup(Solovey, et al 1997)SELPadhesionup(Solovey, et al 1997)SELEadhesionup(Solovey, et al 1997)
Leukocytes
The sickled erythrocytes stimulates leukocyte recruitment: ensuing the inflammatory stimulus, leukocytes are recruited to the activated endothelium of the venous circulation where it forms adhesive interactions with the activated endothelium and sickled erythrocytes, leading to a reduced blood flow and eventually vaso-occlusion (Turhan, et al 2002).
SCD patients characteristically have increased levels of activated leukocytes. It has been demonstrated that SCD-related leukocytes have increased expression and activity of the integrin, beta 2 (complement component 3 receptor 3 and 4 subunit) (ITGB2) LFA-1, integrin, alpha M (complement component 3 receptor 3 subunit) (ITGAM) and integrin, beta 1
(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) ITGB1) in the presence of the inflammatory stimulus, IL-8 (Canalli, et al 2011, Lomakina and Waugh 2010). These SCD leukocyte integrins have been demonstrated to ligate with endothelial ICAM-1 (Canalli, et al 2011) and fibronectin 1 (FN1) (Miguel, et al 2011). The leukocyte recruitment process is enhanced by the overexpression SELP and SELE in endothelial cells as endothelial cells lacking SELP and SELE have demonstrated reduced leukocyte recruitment and diminished vaso-occlusion (Turhan, et al 2002). Additionally, the lack of SELE has also been demonstrated to reduce the adhesion of sickled erythrocytes to leukocytes (Hidalgo, et al 2009).
TABLE 4Leukocyte activation-related molecules in individuals with SCDExpression statusMoleculesTypes of Moleculesin SCDReferencesITGB2leukocyte integrinup(Canalli, et al 2011)ITGAMleukocyte integrinup(Canalli, et al 2011)ITGB1leukocyte integrinup(Canalli, et al 2011)
Platelets
SCD plateles show increased surface expressions of SELP, activated αIIBβ3 (GPIIbIIIa) (Devi, et al 2010) and higher concentrations of the platelet activation markers; platelet factor 4 (PF-4) and β thromboglobulin TGB) (Westwick, et al 1983, Yoong, et al 2003). Activated platelets have been shown to release endothelium activators, such as soluble CD40 ligand (sCD40L) (Lee, et al 2006), PF-4 (Westwick, et al 1983) and IL-1β (Wun, et al 2002). In healthy individuals, platelet adhesion is inhibited by the antithrombotic factor, NO whilst SCD platelet adhesion is stimulated by the activated endothelium releasing ADP, TF, von Willebrand factor (vWF) and the expression of platelet-binding adhesion molecules such as glycoprotein Ib (platelet), beta polypeptide (GPIb), selectin P ligand (SELPLG), vitronectin receptor (VTNR) and ICAM-1 (van Gils, et al 2009). SCD platelets have been demonstrated to have increased adhesion to the αIIbβ3 platelet integrin ligand, fibrinogen that in turn favors the adhesion of platelets to endothelium protein FN1 (Chada, et al 2006). Moreover, platelets and sickled erythrocytes have been demonstrated to aggregate via the formation of thrombospondin bridges thereby contributing to vaso-occlusion (Wun, et al 1999).
TABLE 5Platelet activation-related molecules in individuals with SCDExpressionstatus inMoleculesType of moleculeSCDReferencesαIIbβ3platelet activationup(Devi, et al 2010)PF-4platelet activation/up(Westwick, et al 1983)endothelium activatorsTGBplatelet activationup(Yoong, et al 2003)sCD40Lendothelium activatorsup(Lee, et al 2006)IL-1βendothelium activatorsup(Wun, et al 2002)GPIbadhesionup(van Gils, et al 2009)PSGL-1adhesionup(van Gils, et al 2009)VTNRadhesionup(van Gils, et al 2009)ICAM-1adhesionup(van Gils, et al 2009)fibrinogenplatelet integrin ligandup(Chada, et al 2006)
Hydroxyurea
Hydroxyurea (HU) is a FDA approved drug that is the only current treatment proven to modify the disease process of SCD (Brawley, et al 2008). HU positively counteracts the pathophysiology of SCD by increasing the production of fetal hemoglobin (HbF)-containing erythrocytes via stimulation of the NO-cyclic guanosine monophosphate (cGMP) signaling pathway (Cokic, et al 2003) and indirectly altering gene expression and proteins associated with the pathophysiology of SCD. The increased concentration of HbF-containing erythrocytes dilutes the concentration of sickled erythrocytes, thereby sequentially triggering decreased hemolysis (Olnes, et al 2009), increased NO bioavailability (Conran, et al 2004) and decreased endothelium activation (Haynes, et al 2008), which likely accounts probably accounts for the beneficial effects of HU treatment in SCD patients. However, HU has been demonstrated to reduce leukocyte counts in patients on therapy (Charache, et al 1996). Although HU improved clinical symptoms by reducing pain and vaso-occlusive crises, acute chest syndrome, transfusion requirements, and hospitalization, SCD patients treated with HU have demonstrated side effect such as inducing DNA damage (Friedrisch, et al 2008), reducing sperm counts (Grigg 2007) and producing iron nitrosyl Hb (Lockamy, et al 2003). There is a need in the art for improved SCD therapy that lacks one or more side effects of HU.